Prospect & challenges towards

CRISPR/Cas-based therapy against

ageing-associated laminar deterioration

Vladyslav Vyshnevskyi Petersen

Aarhus University • Faculty of Health

1. June 2022

Abstract
Ageing may be set to become both a demographic and socioeconomic
problem in the future, precipitating the demand for therapies that inhibit
health deterioration due to ageing. In the literature was identified a
gap at the intersection between genetic therapy and the study of the
nuclear lamina, which is involved in genomic regulation and implied in
genetic accelerated-ageing syndromes. The potential for targeting this
using CRISPR/Cas was examined in the form of a literature review.
Primary research involving in vivo therapy, target identification, and the
connection between progeroid pathology and normal ageing was collected
from MEDLINE and through preliminary literature.
While certain mutations in the prelamin A protein are associated
with Hutchinson-Gilford Progeria, it turns out that complete deletion
of the sequence coding for prelamin A leads to a completely healthy
phenotype, while avoiding the range of disorders associated with the
protein. CRISPR/Cas-mediated therapies targeting this mechanism in
conjunction with progeria treatment are well-proven. Furthermore, natural
ageing was shown to be associated with the accumulation of prelamin A
in the nucleoplasm, leading to various degenerations of nuclear integrity
and proteostasis. However, prelamin A was nevertheless found to play a
role with DNA repair in aged cells.
The knock-out of the prelamin A-coding region was proposed as a
plausible high-yield therapy to inhibit natural ageing-associated decline,
though with reservations regarding the need for further knowledge about
the extent to which lamins can replace each other’s functions.

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 Resumé
Aldring har potentiale til at blive et samfundsproblem i den nære
fremtid. Med henblik på dette identificerede man i den videnskabelige
litteratur et mindre hul indenfor genterapi rettet mod cellekernens lamina,
som blandt andet er forbundet med genetiske sygdomme hvor man aldres
betydeligt hurtigere end normalt.
Man anmeldte litteraturen fra forskere der havde prøvet at behandle
sygdommen med genterapi, samt også litteraturen fra forskere som under-
søgte dens årsag og hvad den har til fælles med normal aldring. Denne
information blev brugt til at konkludere at en lignende genterapi rettet
mod normal aldring i cellekernens lamina er mulig at lave.

Excluding tables of contents, abbreviations, and information, as

well as figures and the literature section, 23 849 characters are
written henceforth.

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Contents
Introduction 5
The nuclear lamina is important for (epi)genetic regulation . . . . . . 5
CRISPR/Cas facilitates genetic therapy . . . . . . . . . . . . . . . . . 7

Methodology 8

Results & analysis 9

In vivo therapy of progeria is promising . . . . . . . . . . . . . . . . . 9
Natural ageing and progeroid laminopathy are highly similar . . . . . 12

Discussion 15
Conclusion & perspectives . . . . . . . . . . . . . . . . . . . . . . . . . 16

Literature 17

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Abbrev. Definition

AAV(9) Adeno-Associated Virus (9) one of the most commonly used viral
vectors.

ABE Adenine Base Editor, Cas variant that switches a single base pair
with adenine.

CRISPR Clustered Regularly Interspaced Palindromic Repeats, a genomic

part of the adaptive immune system in prokaryotes.

Cas CRISPR-associated protein, a family of enzymes that can produce

double-strand breaks in DNA at specific loci.

CI Confidence Interval.

gc Genome Copies, an amount of virus or vector particles.

gRNA Guide RNA guides the Cas enzyme to a specific defined locus.

HGPS Hutchinson-Gilford Progeria Syndrome, a genetic disease

characterised by accelerated ageing.

iPSC Induced Pluripotent Stem Cell.

LMNA Gene coding for prelamin A/C.

LMNB1/2 Genes coding for lamin B1 and lamin B2 .

mRNA Messenger RNA, is read by ribosomes and translated into a protein.

mTOR Ageing-associated pathway inhibited by rapamycin.

PCR Polymerase Chain Reaction, a method of polymerising

polynucleotides.

SD Standard Deviation.

SNP Single-Nucleotide Polymorphism, a single-bp gene variation

sgRNA Single Guide RNA, see gRNA

WT Wild-Type, the typical variation of a gene or protein, especially as

it occurs in nature.

VSMC Vascular Smooth Muscle Cell.

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Introduction
Ageing demographics have been predicted to become one of the biggest socioe-
conomic problems in the industrialised world by 2050.1 In Denmark, 19% of
the population is 65 years or older. Nevertheless, this group makes up almost
half of the top 10% of patients that consume 71% of all public health funding.2
Most of these costs are due to complications associated with ageing, as well as
comorbidities exacerbated by the ageing phenotype.
When one examines the field of ageing, one of the standard approaches to
the study of the biology behind what we call ageing is the so-called hallmark
approach, as first proposed by De Grey & Rae (2008) and later formalised in a
landmark review by López-Otín et al. (2013). Different hallmarks define different
pathogenic pathways and stages of ageing.
Among these hallmarks, a sub-area that to date appears to have had relatively
little intersection with genetic engineering using CRISPR/Cas in the academic
ageing research is therapy directed towards the nuclear lamina.

The nuclear lamina is important for (epi)genetic regulation

The nuclear lamina has failed to receive much attention in the literature until
quite recently.3 The lamina is a cytoskeletal layer that envelops the inner
surface of the inner nuclear membrane. It binds and facilitates the stability of
chromatin, both directly or via secondary proteins, and also plays a major role
in euchromatin expression and DNA repair.
Laminar filaments, so-called lamins, fall into two types; those encoded by the
LMNA gene, producing either lamin A or lamin C via differential splicing, and
those encoded by the LMNB1 and LMNB2 genes, resulting in lamin B1 and
lamin B2 respectively. The lamina is also made up of a other proteins with
varying roles, as shown in Figure 1.
Progeria is a laminar disorder characterised by accelerated ageing. The most
famous type is Hutchinson-Gilford progeria syndrome (HGPS). The pathogenesis
typically begins with a sporadic de novo splice-site mutation in exon 11 of the
LMNA gene, which turns the prelamin A protein into a truncated version of
itself, so-named progerin. This results in a severe loss-of-function of the whole
nuclear lamina in its structural, epigenetic and reparatory roles.
Another recently documented progeria is Néstor-Guillermo progeria syndrome
(NGPS), which is caused by a mutation in the laminar BANF1 gene.5 Lastly,
Werner syndrome sets itself apart as being an adult-onset progeria. Typically
1 McMorrow & Roeger (1999)
2 Sundhedsdatastyrelsen (2018)
3 Simpkins (n.d.)
4 Coutinho et al. (2009)
5 Cabanillas et al. (2011)

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Figure 1: The structure of the nuclear lamina. It envelops the inside of the inner nuclear
membrane (INM), to which it is bound via nesprin, emerin, lamina-associated proteins
1 & 2 (LAP1 & LAP2), the lamin B receptor (LMB) and LAM1. It maintains nuclear
stability, chromatin structure and binds nuclear pore complexes (NPC). Transcription
factors bound to the lamina include retinoblastoma transcriptional regulator (RB),
germ cell-less (GCL), sterol response element binding protein (SREBP1), FOS &
MOK2. Barrier to autointegration factor (BAF) and heterochromatin protein (HP1)
aid in holding chromatin in place.4

Figure 2: Truncated prelamin A, called progerin, is unable to be cleaved into lamin A.

HGPS can also less frequently be caused by loss-of-function in the Zmpste24 enzyme.4

6
associated with a mutation in the WRN gene, though a significant subset (15–
20%) is associated with a LMNA mutation instead.6
Because of its role as a regulator of many different hallmarks of ageing, the
nuclear lamina was chosen as a high-yield target for potential ageing therapy.

CRISPR/Cas facilitates genetic therapy

The current gold standard of genetic engineering in regards to both cost, accuracy,
precision and specificity is the use of the Cas family of enzymes, which has
recently enjoyed widespread adoption among researchers. Originally derived from
a part of the anti-viral adaptive immune system in prokaryotes, the technique
relies on the delivery of a so-called Cas-enzyme along with one or more guide
RNA (gRNA) into the nucleoplasm of a cell. The gRNA allows the enzyme to
bind to highly specific loci in the genome and produce a double-strand break at
that location, which can be used to effect knock-out or knock-in of specific genes.
The Cas enzyme, directly or in the form of mRNA, can be packed into viral
vectors, lipid nanoparticles, etc. to achieve delivery into cells in living tissue with
varying degrees of tropism. In addition, the Cas9 enzyme has been engineered
by researchers to be able to specifically edit single base pairs at specific loci,
with or without double-strand breaking.
The objective of this work is to assess the prospect and challenges
of developing a genetic therapy to treat or prevent natural ageing-
associated health decline. This is done on the basis of a literature
review of in vivo CRISPR/Cas-based therapies remedying laminar
progeroid disorders, as well as research linking such progeroid condi-
tions to normal ageing.
In particular, there is an interest in identifying:
• What is the potential efficacy of such an in vivo therapy?
• What potential safety concerns and risks may materialise?
• How can existing methods be applied to natural ageing condi-
tions vis-à-vis pathological ones?

6 Chen et al. (2003)

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Methodology
The literature survey was carried out by searching the MEDLINE database
through the PubMed portal. The overall topic (i.e. the nuclear lamina) was
initially chosen due to the identification of a minor gap in the literature compared
to other hallmarks of ageing. The desired literature in this review is that which
lies within the intersection between studies employing the CRISPR/Cas system,
as well as studies that use various progeria-associated nuclear proteins as targets.
A disjunction was formed between the keywords CRISPR, Cas, Cas9, CRISPR/Cas
and CRISPR/Cas9. This whole disjunction was then conjuncted with the
keywords progeria, Hutchinson-Gilford, Néstor-Guillermo, and (Werner
syndrome[Title/Abstract]) AND (LMNA)).
Most of the returned research was published in the past 3 years, consistent with
the previous observation that the bulk of developments in the sub-field as a
whole are quite recent. Among them, some focused on non-laminar genes as
targets, some were either meta-analytic reviews or news articles, others were
non-therapeutic generations of animal models, and others again only featured the
keywords among citations. Three studies that treated HGPS in vivo were selected
from the results, and a further two studies dealing with target identification were
extracted from a recent review.10
To explicate the connection between laminopathy and normal ageing, one of the
preliminary reviews10 were consulted, and two older articles were identified and
incorporated into the literature corpus. Additionally, a much more recent review
dealing with the lamina and ageing was found in the same journal.10 As well as
citing the older studies from the other review, it also cited newer studies from
the last decade. All of these were incorporated into the final primary corpus.

Category № References & additional notes

In vivo 3 Research evaluating in vivo therapy of laminopathies.7

therapy

Target 2 Studies identifying specific genetic and tissular

identification targets.8

Connection to 4 Group of studies examining the similarities and

normal ageing dissimilarities between laminopathy and ageing.9

Expedient 3 These reviews led to the discovery of the target

reviews identification and ageing-connection studies10

7 Koblan et al. (2021); Beyret et al. (2019); Santiago-Fernández et al. (2019)

8 Fong et al. (2006); Sánchez-López et al. (2021);

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Results & analysis
One of the most striking studies within this sub-field that came to define much
of the study into lamina-directed therapy, including later studies in this review,
was carried out by Fong et al. (2006). The researchers set out to produce a
HGPS mouse model, but via site-directed mutagenesis, the researchers instead
managed to accidentally produce a copy of LMNA with a deletion in exon 11
that did not produce prelamin A but retained expression of lamin C.
The mutated gene was nevertheless used to produce heterozygotic
prelamin Aknock-out mice. These mice were both intrabred with each
other to produce homozygotes and interbred with Zmpste24 -deficient mice and
mice that lacked all of LMNA.
Most strikingly, in terms of typical measures of weight gain and grip strength,
the prelamin A-deficient mice were indistinguishable from their wild-type (WT)
counterparts. In comparison, homozygotically LMNA-deficient mice were ex-
tremely morbose all deceased after 6 weeks. In addition, immunofluorescence
microscopy was used to confirm that all lamina-associated proteins were normally
positioned, in contrast to LMNA-deficient mice.11
However, both homo- and heterozygotic prelamin A-deficient mice showed a
significantly increased prevalence of misshapen nuclei (p < 0.007), despite an
otherwise normal phenotype by all other examined measures. Most interestingly,
this lamin A-deficient allele was shown to be phenotypically dominant; mice
that were heterozygous for lamin A-deficiency but homozygous for Zmpste24 -
deficiency still exhibited a normal phenotype.
This accidental discovery would later set the stage for many studies deliberately
targeting the last two exons of LMNA to achieve a similar effect under various
conditions.

In vivo therapy of progeria is promising

The first time that a CRISPR/Cas-based technique was used in vivo to treat
progeria in a mouse model was by Santiago-Fernández et al. (2019). This study
builds on the previous finding that lamin A appears to be wholly dispensable
for laminar functioning. With this in mind, the researchers produced an sgRNA
that disrupts the expression of exon 11.
The sgRNA was packed inside an adeno-associated virus 9 (AAV9) based vector
along with the Cas9 enzyme and delivered intraperitoneally12 to homozygously
progerin-expressing mice. Due to the tropism of AAV9, succesful indels ranged
9 Scaffidi & Misteli (2006); Ragnauth et al. (2010); Lattanzi et al. (2014); Petrini et al.

(2017)
10 Benedicto et al. (2022); López-Otín et al. (2013); Martins et al. (2020); respectively.
11 This characteristic of abnormally positioned proteins is also a feature of both HGPS and

natural ageing.
12 2 × 1011 genome copies (gc) injected.

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from 13.6 ± 2.6% in liver tissue to 1.1 ± 0.2% in lung tissue, with varying p-values
depending on sex. The measured expression of progerin on a tissue-basis was
completely proportional to the indel success rate in that tissue, although lamin
C increased in all tissues when compared to controls, except for skeletal muscle.
Median survival of the treated mice compared to the controls was increased by
33.5 days, from 127 to 160.5, which is a 26.4% jump. Mean survival jumped
from 128.1 days (SD 15.73; 95% CI 116.8-139.4) to 167.4 days (SD 30.41; 95%
CI 145.6-189.2). Maximum survival jumped from 151 to 212 days (p = 0.0163).
Correspondingly, the treated mice expressed a healthier phenotype, both in terms
of grooming, body weight, and blood glucose. The last metric was significantly
improved in both males (p = 0.0005) and females (p = 0.0206); in both cases
the treated mice showed a near-identical mean value compared to WTs.
An interesting finding in this study is the significant reduction in apoptotic cells
in kidney tissue in treated mice, despite no mutations having been observed in
this tissue. This suggests that the gene therapy had significant systemic effects
outside directly affected cells.

Gene therapy can nevertheless have deleterious consequences

The above study was published immediately before a similar study by Beyret
et al. (2019), in which the researchers procured Cas9 -expressing mice and
crossbred them with mice that heterozygously expressed progerin. The resulting
Cas9 & progerin expressing generation was then in turn intrabred to produce
homozygous progerin-expressing Cas9 mice. Guide RNA (gRNA) targeting the
LMNA locus was packed in AAV9 and intravenuously delivered13 to the neonatal
progerin/Cas9 mice to inhibit both lamin A and progerin expression.
The median survival of homozygous HGPS mice was just under 17 weeks,
in comparison to the gRNA-treated and Cas9 -expressing mice whose median
survival was just over 25 weeks (p < 0.0001). The epidermis and ventricular
mesothelium of the treated mice had almost the same size compared to WTs,
though they both had different histological characteristics, such as increased
adipose tissue in the treated mice. In comparison, the untreated mice showed
advanced degeneration compared to treated (p = 0.0002).
Treated mice ran significantly (p = 0.0457) more revolutions per minute than
untreated mice. They likewise showed significantly improved grip strength
compared to untreated mice (p = 0.0001). In both cases treated mice matched
the performance of WTs.
Unfortunately, 48.7% of the gRNA-treated mice (n = 39) displayed gastroin-
testinal inflammation in the form of megacolon and ultimately succumbed to
excretory dysfunction. All of the remaining mice and all of the negative controls
were asymptomatic.
13 2 × 1010 –1 × 1012 gc “as determined by quantitative PCR”.

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Restoration of prelamin A leads to better rescue of progeria
Two years later, another study (Koblan et al., 2021) set itself apart from the
previous two in its use of a CRISPR-based adenine base editor (ABE) to directly
correct the HGPS-causing SNP and restore normal lamin A production, instead
of eliminating lamin A expression completely. They used a delivery approach
involving a pair of AAVs, each expressing one half of the ABE. To study the
effects of therapy at different stages in the pathology, retro-orbital delivery was
performed in both 3 day- and 14 day-old mice14 expressing the human progerin
mutation. The vector displayed equivalent tropism to the 2019 studies.
Some mice were euthanised at six weeks and six months of age to confirm editing
efficacy. Analysis showed that efficiency ranged from ∼ 60% in liver tissue to
∼ 10% in bone tissue. Mice treated at 14 days of age generally showed higher
editing efficiencies than mice treated at 3 days, which was attributed to the
differing amount of vector particles that could be injected in neonates versus
adolescents.
In this study, the median lifespan of the saline-injected controls was 189 days
for those treated at 3 days old (n = 12) and 215 days for those treated at 14
days old (n = 12). In turn, their base edited counterparts respectively had a
median lifespan of 337 days (80% increase, n = 11, p < 0.0001) and 510 days
(140% increase, n = 10, p < 0.0001). The lattermost value corresponds roughly
to the onset of the gerontological phenotype in WT mice. Furthermore, the mice
treated at 3 days old only had a slightly improved histological phenotype in
cardiovascular tissue, while the mice treated at 14 days old were almost identical
to the WT.
Over 1½ years of longevity studies, 3 out of 48 mice were excluded due to health
conditions deemed unrelated to progeria. Of nine deceased 14-day-old ABE-
treated mice, 1 exhibited gastrointestinal necrosis, 5 exhibited liver tumors and
3 exhibited no apparent abnormalities. Subsequent whole-genome sequencing of
liver tissue revealed that all of the AAV-treated mice showed evidence of rare
cancer-associated AAV integration.
The three studies show that in vivo CRISPR-based therapy that either knocks out
lamin A expression or corrects it is potentially viable and significantly effective
in treating lamin A-associated progeria, improving both lifespan and healthspan.
In particular, correction as opposed to inhibition of lamin A appears preferable.
Nevertheless some highly problematic side effects of vector choice and systemic
gene therapy were identified.

14 5 × 1010 gc and 5 × 1011 gc respectively for each AAV in the pair.

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 Study µ̃LS 15 Cas9 delivery Method

Santiago-Fernández ∼ 27% Intraperitoneal Prelamin A/progerin

et al. (2019) knock-out.

Beyret et al. (2019) ∼ 47% Endogenous Prelamin A/progerin

knock-out.

Koblan et al. (2021) ∼ 80% Retroorbital WT prelamin A

knock-in at 3 days
old.

Koblan et al. (2021) ∼ 140% Retroorbital WT prelamin A

knock-in at 14 days
old.

Therapy can be effective regardless of treatment timing

The last study exemplifies the curious relationship between delivery timing and
therapeutic efficacy. To elucidate this further, a mouse strain that can express
progerin and restore lamin A in a time and cell-type specific manner via Cre
recombinase activation was developed recently by Sánchez-López et al. (2021).
When restoration of lamin A was induced at 6 months of age, the mice would
live 84.5% longer than their control counterparts (p < 0.0001). However, at 13
months of age, the difference was merely 6.7%, though still significant (p < 0.05).
Much more interestingly, when lamin A was selectively restored exclsuively in
vascular smooth muscle cells and cardiomyocytes throughout the whole life of
the mice, they displayed a similar median lifespan to WTs.
This study indicates that genetic therapy targeting LMNA should always be
effective regardless of age, though the effect is inversely correlated to age at
onset. Furthermore, merely targeting cardiovascular myocytes may be enough
to rescue HGPS completely.

Natural ageing and progeroid laminopathy are highly similar

Scaffidi & Misteli (2006) demonstrated for the first time that all of the exact
same physiopathological features that are seen in HGPS occur in completely
normal ageing as well. Cell lines from human donors of all ages were examined
for their expression mutations in exon 11 of LMNA, which were not found
to be significantly more frequent in older individuals (p > 0.1). However, the
same pathological appearance of the nuclear membrane as seen in HGPS, was
15 Median lifespan increase.

12
found with significant frequency in older cells (p < 0.05), in this case due to the
accumulation of WT prelamin A.
The researchers hypothesised that this phenotypical change in older cells was
due to the use of exon 11 of LMNA. When they blocked the expression of this
site using morpholino nucleotides, the older cells immediately and significantly
improved all measured marks of ageing (p < 0.05).

Nuclear deterioration in natural ageing is due to wild-type prelamin

A accumulation
Four years later, Ragnauth et al. (2010) cultured human vascular smooth
muscle cells (VSMC) in vitro in order to elucidate the progeria-ageing connection
further. Like the previous study, they failed to detect significant accumulations
of progerin in healthy aged cells, and they also showed a significant accumulation
of prelamin A in aged cells, compared to young cells. In addition, both senescent
cells and older cells display a propensity for the accumulation of prelamin A,
which is not seen in young healthy cells.
Aged cells were treated with statins in order to inhibit the expression of
prelamin A. This significantly reduced the prevalence of nuclei displaying an
abnormal phenotype in microscopy (p < 0.04), further strengthening the connec-
tion between prelamin A expression and the ageing phenotype. Furthermore,
induction of oxidative stress using H2 O2 was found to increase the expression of
prelamin A.

Paradoxically, prelamin A mediates DNA repair in aged cells

Both of these findings were discussed in (Lattanzi et al., 2014), in which were
examined human fibroblast cell cultures in groups of 8–35 year-old, 65–80 year-
old, as well as 95–105 year old donors. The researchers replicated the same
findings regarding prelamin A and oxidative stress.
Novelly, they found that centenarian cells, which otherwise have the highest
prelamin A levels, appear to have a lower basal level of DNA damage, which
was linked to increased activity of lamina-associated repair enzymes, as well as
chromatin remodelling due to epigenetic effects (p < 0.05).
Fibroblasts were treated with rapamycin, a drug known to extend lifespans
in animal models. To the surprise of the researchers, rapamycin was found to
increase the expression of prelamin A in the cell, which is contrary to the reported
effect of rapamycin on pathological progerin. However, rapamycin was also found
to selectively trigger degradation of farnesylated prelamin A and enhance the
accumulation of defarnesylated lamin A. The study concluded that only the
defarnesylated lamin is associated with desirable epigenetic and genoprotective
properties, and that the accumulation of its farnesylated counterpart was due in
part, but not in whole, to Zmpste24 downregulation with ageing.

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Lamina-associated proteins are mislocated in aged cells
The most recent study (Petrini et al., 2017) examined induced pluripotent
stem cells (iPSC) in vitro. Again, the accumulation of lamin A was found in
older iPSCs. Furthermore, it was established that aged iPSCs are characterised
by increased emerin and nesprin-2 expression, as well as decreased lamin B1
expression (p < 0.05, n = 3).
However, it was also found that the aged cells displayed mislocation of emerin and
nesprin-2 in the cytoplasm and nucleoplasm, as opposed to tight concentration
in the nuclear rim as with younger cells.

Figure 3: Phenotypical differences between the nuclei of young iPSCs (y-iPSC) and
aged iPSCs (a-iPSC), as well as differentiated cells. Emerin and nesprin-2 are encoded
by EMD and SYNE2, respectively. NF-kB is a common biomarker of ageing.

14
Discussion
In terms of efficacy, Koblan et al. (2021) implied that high efficiencies can be
achieved in a dose-dependent manner. Patients in all of the in vivo studies
also displayed dramatic improvements with only a single administration. The
data from Fong et al. (2006) regarding e.g. allele dominance, as well as the
systemic changes observed by Santiago-Fernández et al. (2019), heavily imply
that profound phenotypical changes may be seen with only incomplete changes
in expression. All this together implies that LMNA is an excellent target for
genetic therapy, even with diffuse administration.
All of the selected therapy studies made use of the AAV9 vector for delivery.
As was noted (Koblan et al., 2021) this vector is associated with carcinogenic
integration, and would therefore be unacceptable in a clinical setting, something
characteristic of most viral vectors. The current gold standard for in vivo delivery
to cells is the use of lipid nanoparticles.16 However, these are more expensive to
develop than vectors, and have some safety concerns of their own in regards to
inflammation. Notwithstanding, they were recently deployed on a world-wide
scale during vaccination efforts against SARS-CoV-2 with very imprevalent
complications. A potential clinical therapy should employ this delivery method
or something similar in terms of safety and utility.
When evaluating the literature, it is clear that the genetic and molecular changes
in ageing cells are not completely identical to those associated with e.g. HGPS.
The latter is associated with the overexpression of progerin, while normally aged
cells overexpress non-truncated prelamin A, though progerin expression is also
slightly increased.
The key to the common phenotype appears to be the farnesylation site on the
lamin A protein, encoded in exon 12 of LMNA. In normal post-translation, the
end of the peptide chain of prelamin A is farnesylated, and this same end is then
later cleaved off by Zmpste24 to form lamin A. However, in HGPS, the Zmpste24
recognition site is deleted due to a splice-site mutation, and the farnesyl group
is never cleaved off. (See Figure 2) As Lattanzi et al. (2014) found, ageing cells
tend to accumulate farnesylated copies of prelamin A in part due to lowered
Zmpste24 expression.
This indicates that a knock-out therapy targeting the last two exons of LMNA
may be effective in inhibiting the natural ageing phenotype. However, it was
also shown that the expression of lamin A is important for genomic repair in
high-damage environments. Conversely, it may be possible to achieve the same
result without concern for the loss of lamin A by stimulating the expression
of ZMPSTE24 through mTOR inhibition, as was implied by the rapamycin-
mediated activation of lamin defarnesylation.
Since lamin C -only mice were shown to not express laminar protein mislocation
by Fong et al. (2006), and such mislocation was found to be highly characteristic
16 Yip (2020)

15
of aged cells by Petrini et al. (2017), this brings hope that lamin C may be able
to replace the reparatory role of lamin A. Further evidence along this line of
reasoning was given by Santiago-Fernández et al. (2019), in which it was briefly
mentioned in passing that prelamin A knock-out mice tend to live longer than
WTs, along with a single citation to a somewhat obscure research article that
confirmed this.17

Conclusion & perspectives

All available research evidence suggests that an in vivo CRISPR/Cas-based
therapy for the high-yield treatment and prevention of lamina-associated ageing
is wholly plausible. However, this assertion comes with reservations in regards
to concerns of delivery mechanisms and uncertainy about the importance of
prelamin A expression in high-damage environments.
Nevertheless, LMNA appears to be a particularly advantageous and effective
target for gene therapy, as large desirable phenotypical changes may be observed
with comparatively minor changes in expression. Furthermore, natural and
progeroid laminar ageing happen to share a mechanism that can be remedied
using routes that are well-established in the literature.
Further studies should be carried out to examine the roles of the type A lamins
in regards to DNA repair, and whether lamin C can fully fulfill this function in
isolation.

17 Lopez-Mejia et al. (2014), “The median lifespan of [heterozygotic lamin C ] and [homozy-

gotic lamin C ] mice was about 110 weeks, which was significantly longer than the lifespan of
WT mice (P = 0.0025 and P = 0.0067, respectively)”

16
Literature
Benedicto, I., Chen, X., Bergo, M. O., & Andrés, V. (2022). Progeria: A
perspective on potential drug targets and treatment strategies. Expert Opinion on
Therapeutic Targets, 0 (0), 1–7. https://doi.org/10.1080/14728222.2022.2078699
Beyret, E., Liao, H.-K., Yamamoto, M., Hernandez-Benitez, R., Fu, Y., Erikson,
G., Reddy, P., & Izpisua Belmonte, J. C. (2019). Single-dose CRISPR-Cas9
therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome.
Nature Medicine, 25 (3), 419–422. https://doi.org/10.1038/s41591-019-0343-4
Cabanillas, R., Cadiñanos, J., Villameytide, J. A. F., Pérez, M., Longo, J.,
Richard, J. M., Alvarez, R., Durán, N. S., Illán, R., González, D. J., & López-
Otín, C. (2011). Néstor-Guillermo progeria syndrome: A novel premature
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